The overall objective of this project is to develop strategies, which will allow the efficient and safe[unreadable] treatment of hematopoietic diseases by hematopoietic stem cell (HSC) gene therapy. Unfortunately neither[unreadable] in vitro studies nor studies in the mouse model have been predictive for safety and efficacy of gene transfer[unreadable] in large animals or humans, thus we will use the dog model which will also allow us to evaluate improved[unreadable] strategies directly in a disease model. While gene transfer efficiencies into HSCs in the dog model and other[unreadable] large animals have improved significantly over the past several years, the development of leukemia in 3[unreadable] patients in the French X-linked severe combined immunodeficiency (XSCID) gene therapy trial has shifted[unreadable] the emphasis from efficacy to safety. Thus, we will use the dog model to study the safety of 3 commonly[unreadable] used integrating vector systems: gammaretrovirus, lentivirus, and foamy virus vectors. Using these vector[unreadable] systems, we have been able to achieve efficient transduction of canine long-term repopulating cells with[unreadable] stable gene transfer levels >5% in a significant number of dogs. Thus, we now have a unique resource[unreadable] available to study in Specific Aim 1 the safety of HSC gene transfer with these vectors. In Specific Aim 2, we[unreadable] will further optimize transduction conditions for foamy and lentiviral vectors to minimize risks from insertional[unreadable] mutagenesis. We will focus on lentivirus and foamy virus vectors since, in contrast to gammaretroviral[unreadable] vectors, these vector systems allow for efficient transduction using short transduction cultures. This is[unreadable] particularly important for stem cell gene therapy in a nonmyeloablative transplant setting where[unreadable] maintenance of stem cells is crucial for the ability to compete with surviving endogenous stem cells. Thus, in[unreadable] Specific Aim 3 we will use these vector systems in a nonmyeloablative setting and also explore whether in[unreadable] vivo selection strategies can improve gene transfer levels after nonmyeloablative conditioning. Finally, in[unreadable] Specific Aim 4 we will test improved gene transfer protocols in a canine genetic disease model. The[unreadable] availability of a clinically relevant large animal model should allow us to quickly translate our findings to[unreadable] clinical HSC gene therapy studies.